Lignocellulose is the most abundant biopolymer on earth. Lignocellulose is the major structural component of woody plants and non-woody plants such as grass or straw. Lignocellulosic biomass is a complex substrate in which crystalline cellulose is embedded within a matrix of hemicellulose and lignin. Lignocellulose represents approximately 90% of the dry weight of most plant material with cellulose typically making up between 20% to 50% of the dry weight of lingo-cellulose and hemicellulose typically making up between 20% and 40% of the dry weight of lignocellulose. Large amounts of lignocellulosic residues are produced through forestry, timber and pulp and paper industries and agricultural practices (straw, stover, bagasse, husk, chaff) and many agroindustries. Also municipal waste contain fractions that can be considered as lignocellulose residues, such as paper or cardboard waste, garden waste or waste wood from construction. Due to high abundance and low price lignocellulosic residues are preferred materials for production of biofuels or raw materials thereof, such as lipids. In addition, dedicated woody or herbaceous energy crops with biomass productivity have gained interest as biofuel use.
The production of biofuels, especially ethanol, from lignocellulosic materials by microbial fermentations has been studied extensively. The greatest challenge for utilization of lignocellulosics for microbiological production of biofuels or biofuel feedstocks lays in the complexity of the lignocellulose material and in its resistance to biodegradation. In lignocellulose, cellulose (20-50% of plant dry weight) fibers are embedded in covalently found matrix of hemicellulose (20-40%), pectin (2-20%) and lignin (5-20%) forming very resistant structure for biodegradation. Further, the sugar residues of hemicellulose contain a varying mixture of hexoses (e.g., glucose, mannose and galactose), and pentoses (e.g., xylose and arabinose) depending on the biomass.
Microorganism-based lipids (i.e. single cell oils) can be used as raw materials for production of biofuels such as biodiesel, renewable diesel or bio jet fuel.
The key steps in cellulose degradation and subsequent fermentation into biofuels include the saccharification of the polymeric substrate into simple sugars, usually mediated by the action of at least three enzymes (endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21)) that act in a synergistic manner. These enzymes are usually produced in a dedicated process, representing major expense factor in lignocellulose-based biofuel processes. Simultaneous saccharification and fermentation (SSF) by a single microorganism, also known as consolidated bioprocessing (CBP), is regarded as potential alternative to the dedicated enzyme production by combining both saccharification and biofuel production. Consolidated bioprocessing offers the potential for lower cost and higher efficiency than processes featuring dedicated cellulase production. This can result in avoided costs of capital, substrate and other raw materials, and utilities associated with cellulase production. However, several challenges must be overcome to achieve economically viable production processes, and the maybe most important aspect, given that lipid-producing (oleaginous) organisms are used, is the large quantity of enzyme needed for the efficient hydrolyzation of cellulose, which can be achieved e.g. by genetic engineering. A recent investigation reported on engineered Escherichia coli strains, which were engineered to utilize pretreated lignocellulosic substrates to produce biodiesel, butanol and pinene (Bokinsky et al., 2011).
Rhodococcus opacus strain PD630 is the model oleaginous prokaryote regarding accumulation and biosynthesis of lipids, which serve as carbon and energy storage and can account up to 87% of the cell dry mass in this strain. In wild-type R. opacus PD630 the lipids consist mainly of triacylglycerols and are stored intracellularly. R. opacus has been considered as production strain for triacylglycerols (TAGs) from renewable resources for the production of biodiesel, monoalkyl esters of short chain alcohols and long chain fatty acids, due to its high substrate tolerance, high density culturing and rapid growth, which make it favorable over other production organisms. Unfortunately, in contrast to other Rhodococcus strains like R. erythropolis, R. opacus PD630 does not use cellobiose (1,4-β-D-glucopyranosyl-D-glucopyranose), the main product of extracellular bacterial and fungal cellulases, as sole carbon and energy source. Genetic analysis suggested that this inability is caused by the lack of a β-glucosidase, rendering the strain unable to hydrolyze cellobiose into its glucose monomers.
WO2011/163348 discloses an integrated suite of process to make jet fuel from wood. Publication proposes [paragraph 0037] that tri-acylglycerol could be produced by fermenting cellulose or hemicellulose from wood with lipid accumulating microbes such as algae or bacteria. However, none of the disclosed embodiments uses cellulosic substrates as a carbon source in fermentation.
Production of oil from lignocellulosic residue materials by microorganisms is attractive from sustainability point-of-view. Although lignocellulose is the most abundant plant material resource, its usability has been curtailed by its rigid structure. In addition to effective pretreatment also reasonably costly enzymatic hydrolysis is required before oleaginous cells could utilize cellulosic substrates. Thus there is a need for oleaginous bacterial strains that could utilize cellulosic substrate at least to some extent. There is also a need for methods of producing lipids on cellulosic substrate. This invention meets these needs.